Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disease characterized by progressive loss of motoneurons and degeneration of motor axons, which results in motor dysfunction and shortening of life span. About 15% of ALS patients are patients with familial ALS (FALS), and about 15% to 25% of FALS patients carry mutations in the gene encoding Cu2+/Zn2+ superoxide dismutase (SOD1). Transgenic mice with high levels of mutant SOD1 protein and activity develop diseases similar to both familial and sporadic ALS, and such muted SOD1 overexpressing transgenic mice are used as a model for ALS. In the invention, too, G93A mice (Science, 264, 1772-1775, 1994) were used as muted SOD1 (G93A) overexpressing transgenic mice.
Since degeneration of motoneurons is thought to be a first sign of the disease, many approaches had been focused to directly support the survival of motoneurons. However, these attempts were not satisfactory.
On the other hand, Brujin et al. reported that prominent SOD1 containing inclusions in astrocytes appear prior to clinical signs and increase markedly in abundance during disease progression, implicating astrocytes as primary targets for mutant-SOD1 mediated damage in mutant SOD1 (G85R) overexpressing transgenic mice (Neuron, 18, 327-338, 1997). Therefore, a bifunctional substance that can restore the function of astrocytes and directly promote motoneurons from each cell death would be more ideal for the treatment of ALS. Such substance, however, has not been known yet.
Hepatocyte growth factor (HGF) was first identified as a potent mitogen for mature hepatocytes, and its gene was cloned in 1989 (Biochem. Biophys. Res. Commun., 122, 1450-1459, 1984; Nature, 342, 440-443, 1989). Although HGF was discovered as a hepatotrophic factor, recent extensive studies on expression and functional analysis including knockout/in mice strategies revealed HGF as a new neurotrophic factor (Ciba. Found. Symp., 212, 198-211, 1997; Nat. Neurosci., 2, 213-217, 1999). HGF shows neurotrophic activities on hippocampus, cerebral cortex, midbrain dopaminergic, cerebellum granular, sensory and motoneurons and sympathetic neuroblasts (Ciba. Found. Symp., 212, 198-211, 1997; Brain Res. Mol. Brain Res., 32, 197-210, 1995). Especially, HGF is shown to be one of the most potent survival promoting factors for motoneurons comparable with glial cell line derived neurotrophic factors (GDNF) in vitro (Neuron, 17, 1157-1172, 1996). Neurotrophic effects of HGF on embryonic spinal motor neurons during development and on adult motor neurons after axotomy of hypoglossal nerve are shown in vivo (J. Neurosci., 20, 326-337, 2000; Eur. J. Neurosci., 11, 4139-4144, 1999). However, nothing has been reported about the role of HGF in ALS, or clinical effect of expression of HGF on ALS, and therefore it has been known whether HGF would be a remedy for ALS or not.
In this background, the present inventors investigated into possibility of using HGF as a remedy for ALS in order to present a novel remedy for ALS.
The inventors first studied the role of HGF in ALS by using G93A transgenic mice as a model for ALS. As a result, it is found that c-Met/HGF receptor-like immunoreactivity (c-Met-IR) is localized in motoneurons of G93A transgenic mice similarly to that of wildtype littermates. Quantification of the levels of the expression of c-met and HGF mRNA in the ventral horn of spinal cord (where motoneurons locate) revealed that they were progressively increased during the progression of ALS in G93A transgenic mice. These results suggested the roles of HGF on ALS motoneurons.
Using neuron-specific enolase promoter (NSE), the inventors generated transgenic mice overexpressing rat HGF in a neuron-specific manner, and explored the effect of HGF on ALS. As a result, it was discovered for the first time that HGF is effective to attenuate motoneuronal death and suppress axonal degeneration of motoneurons. It was also found that HGF exerts a neuroprotective effect not only on motoneurons but also on DRG sensory neurons against ALS related neurotoxicity. The neuroprotective effect of HGF was also indicated from the delayed loss of muscle weight in ALS.
The inventors further studied if the progress of ALS can be actually suppressed (delayed) by HGF or not by using the same transgenic mice. To a great surprise, start of paralysis was delayed, the life span was extended, and motor functions were improved by an extremely small expression of HGF (about 2 times of wildtype mouse and G93A mouse).
On the basis of these findings, it was discovered for the first time that the HGF has a therapeutic effect on ALS.
The inventors further investigated into the mechanism of suppressing progress of ALS by HGF, and revealed that HGF brings about the ALS improving effects by at least three new mechanisms as explained below.
(1) Induction Suppressing Action of Caspase-1 on Motoneurons
At middle stage of ALS, caspase-1 is thought to play an important role in the progress of the disease, because caspase-1 is shown to be activated and/or induced in motoneurons of transgenic mice overexpressing mutated SOD1 (Proc. Natl. Acad. Sci. U.S.A., 95, 15763-15768, 1998; Science, 288, 335-339, 2000), and introduction of dominant negative inhibitor for caspase-1 in G93A mice was successfully delayed in mortality for about 2 weeks (Nature, 388, 31, 1997; J. Exp. Med., 185, 933-940, 1997). Accordingly, the inventors examined whether HGF can modify the induction of caspase-1, and disclosed that HGF has an effect of suppressing induction of caspase-1 in ALS motoneurons.
(2) Phosphorvylation of Akt in Spinal Cord
Phosphorylation of Akt is suggested to be involved in the survival promoting activity of HGF in cerebral cortex neurons and kidney epithelial cells, and HGF is shown to induce Bcl-xl expression and block massive apoptosis in the liver in fluminant hepatitis models (Biochem. Biophys. Res. Commun., 244, 683-690, 1998; Hepatology, 30, 151-159, 1999). Accordingly, the present inventors explored if phosphorylation of Akt would be induced by expression of HGF, and recognized phosphorylation of Akt specifically in the spinal cord of ALS.
(3) Suppression of Down-regulation of Glial-specific Glutamate Transporter (EAAT2/GLT1) in Reactive Astrocytes
It has been indicated that glutamate-mediated excitotoxicity contributes to motor neuron degeneration of ALS by reduction of glutamate clearance.
Consistently with this hypothesis, it is reported that in the spinal cord and motor cortex of patients with sporadic ALS, glutamate transport activity is decreased remarkably (N. Engl. J. Med., 326, 1464-1468, 1992), and immunoreactivity for glial-specific glutamate transporter (EAAT2/GLT-1), which locates in astrocytes and is thought to be a major transporter to suppress glutamatergic neurotoxicity, selectively disappears (Ann. Neurol., 38, 73-84, 1995). The reduction of EAAT2 in G85R type ALS model transgenic mice (Neuron, 18, 327-338, 1997), and SOD1 mutants (A4V and I113T)-linked inactivation of a glial glutamate transporter in ALS (Nat. Neurosci., 2, 427-433, 1999) are also reported. The inventors previously studied the role of HGF on EAAT2 because glutamatergic neurotoxicity was thought to play a part in motoneuron degeneration in ALS. As a result, it was found that HGF functions to suppress down-regulation of EAAT2 in ALS and maintain a functional astrocytes in endstage of ALS.
It was thus disclosed that HGF improves ALS by at least three mechanisms, that is, induction suppressing action of caspase −1 on motoneurons, phosphorylation of Akt, and suppression of down-regulation of glial-specific glutamate transporter (EAAT2/GLT1) in reactive astrocytes.
Thus, HGF has an effect of improving the motor function of ALS and life span through two actions, that is, direct neuronutrient factor activity on motoneurons, and indirect improving action of glutamate cytotoxicity on motoneurons by maintaining the level of glutamate transporter in astrocytes. Such bifunctional growth factor has not bee known so far, and HGF is a first example. Such functions of HGF suggest possibility therapeutic usefulness of HGF (gene or protein) in ALS and related motoneuron diseases.